首页> 外文期刊>Investigative ophthalmology & visual science >Retinol-binding site in interphotoreceptor retinoid-binding protein (IRBP): a novel hydrophobic cavity.
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Retinol-binding site in interphotoreceptor retinoid-binding protein (IRBP): a novel hydrophobic cavity.

机译:受体间类视黄醇结合蛋白(IRBP)中的视黄醇结合位点:一种新型的疏水腔。

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PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) appears to target and protect retinoids during the visual cycle. X-ray crystallographic studies had noted a betabetaalpha-spiral fold shared with crotonases and C-terminal protein transferases. The shallow cleft formed by the fold was assumed to represent the retinol-binding site. However, a second hydrophobic site consisting of a highly restricted cavity was more recently appreciated during in silico ligand-docking studies. In this study, the ligand-binding environment within the second module of Xenopus IRBP (X2IRBP) is defined. METHODS: Pristine recombinant polypeptide corresponding to X2IRBP was expressed in a soluble form and purified to homogeneity without its fusion tag. Phenylalanine was substituted for tryptophan at each of the putative retinol-binding domains (W450F, hydrophobic cavity; W587F, shallow cleft). Binding of 11-cis and all-trans retinol were observed in titrations monitoring retinol fluorescence enhancement, quenching of tryptophan fluorescence, and energy transfer. The effect of oleic acid on retinol binding was also examined. RESULTS: A ligand-binding stoichiometry of approximately 1:1 was observed for 11-cis and all-trans with K(d) in the tens of nanomolar range. The substitution mutants showed little effect on retinol binding in titrations after fluorescence enhancement. However, the W450F and not the W587F mutant showed a markedly reduced capacity for fluorescence quenching for both 11-cis and all-trans retinol. Oleic acid inhibited the binding of 11-cis and all-trans retinol in an apparent noncompetitive manner. CONCLUSIONS: The binding site for 11-cis and all-trans retinol is a novel hydrophobic cavity that is highly restrictive and probably distinct from the long chain fatty acid-binding site.
机译:目的:感光细胞间的类视黄醇结合蛋白(IRBP)在视觉周期中似乎靶向并保护类视黄醇。 X射线晶体学研究已注意到与巴豆酶和C端蛋白转移酶共有一个betabetaalpha螺旋折叠。褶皱形成的浅裂被认为代表视黄醇结合位点。然而,在计算机配体对接研究中,最近发现由高度受限的空腔组成的第二个疏水位点。在这项研究中,定义了非洲爪蟾IRBP(X2IRBP)第二个模块内的配体结合环境。方法:以可溶形式表达与X2IRBP对应的原始重组多肽,并纯化至均质而无其融合标签。在每个假定的视黄醇结合域(W450F,疏水腔; W587F,浅裂)处,苯丙氨酸代替色氨酸。在监测视黄醇荧光增强,色氨酸荧光猝灭和能量转移的滴定中观察到了11-顺式和全反式视黄醇的结合。还检查了油酸对视黄醇结合的影响。结果:观察到大约1:1的配体结合化学计量比,在K的(d)在几十纳摩尔范围内的11-顺式和全反式。取代突变体在荧光增强后的滴定中对视黄醇结合几乎没有影响。但是,W450F而不是W587F突变体显示11-顺式和全反式视黄醇的荧光猝灭能力明显降低。油酸以明显的非竞争性方式抑制11-顺式和全反式视黄醇的结合。结论:11-顺式和全反式视黄醇的结合位点是一个新的疏水腔,具有高度的限制性,可能与长链脂肪酸结合位点不同。

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